Ambion turbo dna free

These protocols have been described earlier (Awasthi et al. 2016a ). Briefly, RNA was isolated from frozen plant samples, using TRIzol ® (Invitrogen, Life Technologies, USA) and quantified using a spectrophotometer (Thermo Scientific, USA). Post DNase (DNA-free™ kit; Ambion ® TURBO DNA-free™, Life Technologies, USA) treatment of RNA, cDNA was synthesized with ImProm-II™ Reverse Transcription System (Promega, USA) using oligo-dT 12 primer (FirstChoice ® RLM-RACE Kit, Ambion ® , Life Technologies, USA). Degenerate primers were used for amplification of the core region (internal fragment) of the gene. This fragment was geleluted (Qiaex II gel extraction kit, Qiagen, Germany) and sequenced. The sequence was used for designing gene Table1 specific primers. First Choice ® RLM-RACE kit (Invitrogen, USA) was used for performing Rapid amplification of cDNA ends (RACE) reactions to clone CHS from D. gotadhora (DbCHS), following manufacturer's instructions. 2 µg of DNase treated RNA, isolated from the leaf tissue of D. gotadhora, served as template for first strand cDNA synthesis. 3′ RACE-PCR was carried out using oligo(dT) primed cDNA and 3′ RACE outer primer along with 3′ RACE-Gene specific primer (GSP). Similarly, 5′ end of the gene was generated using 5′ RACE outer primer and 5′ RACE GSP along with 5′ RACE inner primer. The 5′ and 3′ RACE amplicons were cloned and sequenced.